Preliminary Analysis Of AChR-Fc Fusion Protein To Myasthenia Gravis By Targeting BCR

Myasthenia gravis is an autoimmune disease mediated by autoantibody, participated by T cells and dependent by complement. It has provided a model for studying other antibody-mediated autoimmune disorders, because the pathogenic target as well as antuantibody is clearly defined and animal model is easy to be constructed. Acetylcholine receptor (AChR) at the neuromuscular junction is the binding target of pathogenic autoantibody. This in combination with complement results in the loss of the AChR and skeletal muscle weakness, which can be life-threatening when respiratory muscle is involved. Current treatments mainly include acetylcholinesterase inhibitors and immunosuppresants, but the radical cure still can't be achieved. Therefore, investigators are searching a more efficient and specific therapy.In recent years, data have emerged suggesting that B lymphocytes play a broader role in immune responses and are not merely the passive recipients of signals that result in the production of antibody. Along with their traditional roles as precursors of antibody-producing plasma cells, B cells have also been found to present antigen, regulate antigen presenting cells (APCs) and T cell functions, produce cytokines, and express receptor/ligand pairs that previously had been thought to be restricted to other cell types. The role of pathogenic B cells becomes more important and complex when the breakdown of self-tolerance, especially in humoral-mediated autoimmune disease. Therefore, therapies that target B cells may be a valuable therapeutic approach for many autoimmune diseases.Considering the critical role of AChRAb in MG, it will be possible to cure the disease if we can eliminate the root cause-AChRAb producing B cells. However, there is no strategy of specifically eliminating AChR reactive B cells to date. Our aim was to design a protein that consists of the extracellular domain of AChRαsubunit (Hα1–210) and the CH2 and CH3 domains of the human IgG1 heavy chain. This fusion protein is expected to selectively kill AChR-specific B cells by cross-linking the B cell receptor (BCR) that has an identical specificity with that of the secreted antibody and inhibitory receptor FcγRⅡB on the surface of B cells. Besides that, Fc fragment can bind Fc receptor of mononuclear macrophage and complements, and mediate ADCC and cell lysis function. We detected the binding ability and cytotoxicity of AChR-Fc fusion protein in an AChRAb-reactive hybridoma cell line in vitro. In additon, we construct EAMG by immunized Lewis rat with ECD protein and study the effects of fusion protein to AChR reactive B cells in MG.1. Expression, purification and characterization of AChR-Fc fusion proteinThe extracellular domain of the human AChRα1-subunit (Hα1-210) was amplified by PCR with the plasmid of AChRα1-subunit as template and then inser ted into pAN1782 mammalian expression vector with a cytomegalovirus promoter, a murine immunoglobulinκ-chain leader sequence and the human genomic IgGγ1 constant region from the hinge to the end of CH3. Recombinate expression plasmid was constructed and transient transfected into CHO-K1 cells. ELISA and Western blot analysis demonstrated the expression of AChR-Fc fusion protein in the culture supernatant. The CHO-K1 cells stably expressing AChR-Fc fusion protein were obtained after selecting by G418. Then the AChR-Fc fusion protein was purified by Protein A affinity chromatograph method. SDS-PAGE further confirmed the expression and purity of the purified protein.2. In vitro binding ability and cytotoxicity assay of AChR-Fc fusion protein on hybridoma cellsTIB175 hybridoma cells were cultivated with complex medium RPMI-1640 supplemented with 10% (v/v) heat-inactivated FCS. Hybridoma cell surface were examined to express BCR and FcγRⅡB by using FITC-conjugated mouse anti-rat CD32 and monoclonal anti-ratκ+λlight chains Ab via flow cytometry analysis. Hybridoma cells were incubated with AChR-Fc fusion protein, FCM analysis showed AChR-Fc fusion protein showed a higher binding capacity to hybridoma cells using FITC-anti humanγchain mAb.The hybridoma cells were incubated with serial dilutions of AChR-Fc or human IgG at different concentrations: 100μg/ml, 50μg/ml, 10μg/ml and medium alone. After 48 h incubation, cell numbers count demonstrated the rate of vial cells was decreased with a dose-dependent way in fusion protein-treated group; Cells were simultaneously double stained with annexin V-?uorescein isothiocyanate (FITC) and PI. FCM analysis showed AChR-Fc can induce the apoptosis of hybridoma cells. Tunel staining and laser scanning confocal 11 microscope (LSCM) further detected the apoptosis; BrdU was added at 16 h before cell harvesting, and at the end of incubation FCM analysis demonstrated BrdU positive cells in fusion protein-treated group were lower than that of other groups. The result of cell cycle showed fusion protein can inhibit the proliferation of cells by G1 phase blockage. In addition, peripheral blood mononuclear cell (PBMC) as effector cells and complement as effector molecule were added to co-incubation system of fusion protein and hybridoma cells, FCM analysis demonstrated fusion protein facilitated PBMC and complement-induced cell apoptosis through Fc region.3. The effects of AChR-Fc fusion protein to AChR reactive B cells in EAMG animal modelRecombinant expression ECD protein of human muscle AChR, emulsified in complete Freund's adjuvant immunized female Lewis rats to induce EAMG animal model. Spleen cells were isolated and incubated with AChR-Fc fusion protein in vitro. ELISPOT assay showed incubation with AChR-Fc fusion protein resulted in a dose-dependent reduction in spleen cells of EAMG. FCM analysis of spleen cells after incubation with fusion protein for 48h showed fusion protein can lead to the apoptosis of CD19+B cells. Rats were treated with AChR-Fc fusion protein or human IgG or PBS by vena caudalis administration for 4 weeks, 3 times per week. Sera were obtained every week and detected by ELISA assay. Treatment with fusion protein resulted in a sharp reduction of level of IgG anti-AChR. ELISPOT analysis showed reduction in the number of AChR-reactive B cells in AChR-Fc treatment Splenocytes from EAMG rats.Our study expressed successfully AChR-Fc fusion protein and analyzed the effects of the fusion protein to AChR reactive B cells through in vivo as well as in vitro study, which may lay a foundation to further study the efficacy of the fusion protein to MG. Such a new strategy that target elimination autoreactive B cells may provide a platform for the treatment of autoimmune diseases.